Surface plasmon sensor

ABSTRACT

A surface plasmon sensor comprises a prism, a metal film, which is formed on one surface of the prism and is brought into contact with a sample, and a light source for producing a light beam. An optical system causes the light beam to pass through the prism and to impinge upon an interface between the prism and the metal film such that various different angles of incidence may be obtained with respect to the interface. A photodetector detects an intensity of the light beam, which has been totally reflected from the interface, with respect to each of the various different angles of incidence. An electrode stands facing the metal film with a liquid sample intervening therebetween, and a DC voltage is applied across the electrode and the metal film. A substance contained in the liquid sample is thus analyzed quickly and with a high sensitivity.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a surface plasmon sensor for quantitativelyanalyzing a substance in a sample by utilizing the occurrence of surfaceplasmon. This invention also relates to an evanescent ellipsosensor,wherein a light beam impinging upon a prism is totally reflected from aninterface between the prism and a sample, a change in condition ofpolarization of the light beam due to the total reflection is detected,and a substance in the sample is thereby analyzed. This inventionfurther relates to an electrophoresis sensor for analyzing a substancein a sample by utilizing electrophoresis.

2. Description of the Prior Art

In metals, free electrons vibrate collectively, and a compression wavereferred to as a plasma wave is thereby produced. The compression waveoccurring on the metal surface and having been quantized is referred toas the surface plasmon.

Various surface plasmon sensors for quantitatively analyzing a substancein a sample by utilizing a phenomenon, in which the surface plasmon isexcited by a light wave, have heretofore been proposed. As one of wellknown surface plasmon sensors, a surface plasmon sensor utilizing asystem referred to as the Kretschman arrangement may be mentioned. Thesurface plasmon sensor utilizing the system referred to as theKretschman arrangement is described in, for example, Japanese UnexaminedPatent Publication No. 6(1994)-167443.

Basically, the surface plasmon sensor utilizing the system referred toas the Kretschman arrangement comprises (i) a prism, (ii) a metal film,which is formed on one surface of the prism and is brought into contactwith a sample, (iii) a light source for producing a light beam, (iv) anoptical system for causing the light beam to pass through the prism andto impinge upon the interface between the prism and the metal film suchthat various different angles of incidence may be obtained with respectto the interface, and (v) a photo detecting means capable of detectingthe intensity of the light beam, which has been totally reflected fromthe interface, with respect to each of the various different angles ofincidence.

In order for various different angles of incidence to be obtained, alight beam having a comparatively small beam diameter may be deflectedand caused to impinge upon the interface. Alternatively, a light beamhaving a comparatively large beam diameter may be converged on theinterface such that the light beam may contain components, which impingeat various different angles of incidence upon the interface. In theformer case, the light beam, which comes from the interface at variousdifferent angles of exit in accordance with the deflection of theincident light beam, may be detected with a small photodetector, whichmoves in synchronization with the deflection of the light beam, or maybe detected with an area sensor extending in the direction, along whichthe angle of exit of the light beam changes. In the latter case, thelight beam may be detected with an area sensor extending in a directionsuch that the area sensor can receive all of the light beam componentscoming from the interface at various different angles of exit.

With the surface plasmon sensor having the aforesaid constitution, whena light beam composed of a P-polarized light component (i.e., apolarized light component normal to the reflection interface) impingesat a specific angle of incidence θ_(SP), which is not smaller than thetotal reflection angle, upon the metal film, an evanescent wave havingan electric field distribution occurs in the sample, which is in contactwith the metal film, and the surface plasmon is excited at the interfacebetween the metal film and the sample by the evanescent wave. In caseswhere the wave vector of the evanescent wave coincides with the wavenumber of the surface plasmon and wave number matching is obtained, theevanescent wave and the surface plasmon resonate, and energy of thelight transfers to the surface plasmon. As a result, the intensity ofthe light, which is totally reflected from the interface between theprism and the metal film, becomes markedly low.

If the wave number of the surface plasmon is found from the specificangle of incidence θ_(SP), at which the aforesaid phenomenon occurs, adielectric constant of the sample can be calculated. Specifically, theformula shown below obtains. ##EQU1## wherein K_(SP) represents the wavenumber of the surface plasmon, ω represents the angular frequency of thesurface plasmon, c represents the light velocity in a vacuum, ε_(m)represents the dielectric constant of the metal, and ε_(S) representsthe dielectric constant of the sample.

If the dielectric constant ε_(S) of the sample is found, theconcentration of a specific substance contained in the sample can becalculated from a predetermined calibration curve, or the like.Therefore, the specific substance contained in the sample can bequantitatively analyzed by finding the specific angle of incidenceθ_(SP), at which the intensity of the reflected light beam becomes low.

However, with the conventional surface plasmon sensors described above,the problems are encountered in that, when the substance contained in atrace amount in the liquid sample is analyzed, the sensitivity withwhich the substance to be analyzed is detected cannot be kept high, anda long time is required to carry out the analysis.

Also, it has heretofore been known that, when a light beam traveling ina first medium is totally reflected by an interface between the firstmedium and a second medium, which has a refractive index lower than therefractive index of the first medium, light referred to as theevanescent wave leaks to the second medium. When the light beam impingesupon the interface, the electric field of light changes in phase beforethe light beam is totally reflected from the interface and after thelight beam is totally reflected from the interface. The change in phasevaries for the P-polarized light component (normal to the reflectioninterface) and the S-polarized light component (parallel to thereflection interface). The change in the condition of polarization isinherent in accordance with the second medium which interacts with theevanescent wave.

Evanescent ellipsosensors for analyzing a substance contained in asample by utilizing the phenomenon described above have heretofore beenused. In the evanescent ellipsosensors, a constitution for totallyreflecting a light beam from an interface between the sample and a prismis employed, and a technique (ellipsometry) for detecting a change inphase difference, i.e. a change in condition of polarization, is appliedto the constitution. Such an evanescent ellipsosensor is described in,for example, PHYSICAL REVIEW LETTERS, Vol. 57, No. 24, 15 December,1986, pp. 3065-3068. With the evanescent ellipsosensors, the prism isemployed as the aforesaid first medium, the sample serving as theaforesaid second medium is brought into close contact with one surfaceof the prism, and the light beam is totally reflected from the interfacebetween the prism and the sample. A change in condition of polarizationdue to the total reflection is detected, and physical properties or atotal amount of the substance in the sample is thereby determined.

However, with the conventional evanescent ellipsosensors describedabove, the problems are encountered in that, when the substancecontained in a trace amount in the liquid sample is analyzed, thesensitivity with which the substance to be analyzed is detected cannotbe kept high, and a long time is required to carry out the analysis.

Further, electrophoresis apparatuses for analyzing substances containedin a sample by utilizing electrophoresis have heretofore been used. Suchan electrophoresis apparatus is described in, for example, JapaneseUnexamined Patent Publication No. 62(1987)-220853. Basically, with theelectrophoresis apparatuses, a DC voltage is applied across anelectrophoresis medium having been impregnated with a sample, andsubstances having electric charges, such as protein, proteindecomposition products, nucleic acid, and nucleic acid decompositionproducts, which substances are contained in the sample, are therebycaused to migrate through the electrophoresis medium. The substances arethus separated spatially in the electrophoresis medium by theutilization of differences in migration speed among the substances.

In the conventional electrophoresis apparatuses, ordinarily, gel sheetsconstituted of apolyacrylamide gel, an agarose gel, or the like, areemployed as the electrophoresis media. Also, ordinarily, in order forthe spatially separated substances to be analyzed, techniques areemployed wherein the substances in the sample are labeled withfluorescent substances, radioactive isotopes, and the like, and thepositions of the labeled substances after migrating through theelectrophoresis medium are recorded on a photographic material, astimulable phosphor sheet described in Japanese Unexamined PatentPublication No. 62(1987)-90600, or the like.

However, in order for the positions of the labeled substances to berecorded, it is necessary to carry out exposure of the photographicmaterial, the stimulable phosphor sheet, or the like, development of alatent image on the photographic material, a process for reading outimage information having been recorded on the stimulable phosphor sheet,and the like. Therefore, with the conventional electrophoresisapparatus, considerable time and labor are required to analyze thesamples. Also, considerable time and labor are required to label thesubstances in the sample with radioactive isotopes, or the like.Further, there is the risk that the labeling operation adversely affectthe human body.

SUMMARY OF THE INVENTION

The primary object of the present invention is to provide a surfaceplasmon sensor, with which a substance contained in a liquid sample isanalyzed quickly and with a high sensitivity.

Another object of the present invention is to provide a surface plasmonsensor, wherein multiple reflection interference of light does notoccur, and the size of an optical system is kept small.

A further object of the present invention is to provide an evanescentellipsosensor, with which a substance contained in a liquid sample isanalyzed quickly and with a high sensitivity.

A still further object of the present invention is to provide anelectrophoresis sensor, wherein complicated operation and process neednot be carried out, and a substance contained in a sample is analyzedsimply.

The present invention provides a first surface plasmon sensor,comprising:

i) a prism,

ii) a metal film, which is formed on one surface of the prism and isbrought into contact with a sample,

iii) a light source for producing a light beam,

iv) an optical system for causing the light beam to pass through theprism and to impinge upon an interface between the prism and the metalfilm such that various different angles of incidence may be obtainedwith respect to the interface, and

v) a photo detecting means capable of detecting an intensity of thelight beam, which has been totally reflected from the interface, withrespect to each of the various different angles of incidence,

wherein the improvement comprises the provision of:

a) an electrode, which stands facing the metal film with a liquid sampleintervening therebetween, and

b) means for applying a DC voltage across the electrode and the metalfilm.

The first surface plasmon sensor in accordance with the presentinvention may be modified such that the sample analysis can also becarried out on the side of the electrode which is provided for voltageapplication. Specifically, the present invention also provides a secondsurface plasmon sensor, wherein the aforesaid first surface plasmonsensor in accordance with the present invention is modified such that itmay further comprise:

a second prism, the electrode being formed on one surface of the secondprism and constituted of a metal film,

a second light source for producing a second light beam,

a second optical system for causing the second light beam to passthrough the second prism and to impinge upon an interface between thesecond prism and the electrode such that various different angles ofincidence may be obtained with respect to the interface between thesecond prism and the electrode, and

a second photo detecting means capable of detecting an intensity of thesecond light beam, which has been totally reflected from the interfacebetween the second prism and the electrode, with respect to each of thevarious different angles of incidence.

The present invention further provides a third surface plasmon sensorwherein, in lieu of the electrode for voltage application being locatedsuch that it may stand facing the metal film as in the aforesaid firstsurface plasmon sensor in accordance with the present invention, theelectrode for voltage application is located at a position spaced apartfrom the metal film on the one surface of the prism.

The present invention still further provides fourth, fifth, and sixthsurface plasmon sensors wherein, in lieu of the metal film being broughtinto direct contact with the liquid sample as in the aforesaid first,second, and third surface plasmon sensors in accordance with the presentinvention, respectively, a sensor film, to which an antigen or anantibody capable of undergoing an antigen-antibody reaction with asubstance to be analyzed, that is contained in a liquid sample, has beenfixed, is formed on the metal film and is brought into contact with theliquid sample.

The present invention also provides a seventh surface plasmon sensor,comprising:

i) a substrate permeable to light,

ii) a light source for producing a light beam, which impinges upon thesubstrate,

iii) an optical system for causing the light beam to impinge upon thesubstrate,

iv) a grating for entry of the light beam, which grating is formed at aportion of one surface of the substrate on the light beam incidenceside, the grating diffracting the light beam and entering the light beaminto the substrate,

v) a sensor means, which is formed on the other surface of the substrateopposite to the one surface and is constituted of a metal film, that isbrought into contact with a sample,

vi) a grating for radiation of the light beam, which grating is formedat a different portion of the one surface of the substrate on the lightbeam incidence side, the grating diffracting the light beam, which hasbeen reflected from an interface between the substrate and the sensormeans, and radiating the light beam out of the substrate, and

vii) a photo detecting means capable of detecting an intensity of thelight beam, which has been radiated out of the substrate,

whereby a wave number of a surface plasmon, which occurs at an interfacebetween the sensor means and the sample, is specified from the intensityof the light beam having been detected by the photo detecting means, anda substance contained in the sample is analyzed quantitatively.

In the seventh surface plasmon sensor, the light beam having beenproduced by the light source is irradiated by the optical system to thesubstrate and is diffracted by the grating for entry of the light beam.The diffracted light beam passes through the substrate and impinges uponthe interface between the sensor means and the substrate.

Also, in the seventh surface plasmon sensor, the substrate permeable tolight may be constituted of, for example, a glass substrate. Thesubstrate may be constituted of a single piece. Alternatively, thesubstrate may be constituted of a plurality of pieces. In the lattercase, for example, the substrate may be constituted of a glass plate, onwhich the grating for entry of the light beam and the grating forradiation of the light beam are formed, and a glass plate, on which thesensor means is formed, the two glass plates being combined with eachother with a refractive index matching liquid intervening therebetween.

The present invention further provides a first evanescent ellipsosensor,comprising:

i) a prism,

ii) a transparent electrode, which is formed on one surface of the prismand is brought into contact with a liquid sample,

iii) a beam irradiating system for irradiating a light beam, which hasbeen polarized in a predetermined condition of polarization, from theside of the prism such that the light beam may be totally reflected froman interface between the liquid sample and the transparent electrode,

iv) means for detecting a change in condition of polarization of thelight beam due to the total reflection,

v) an opposite electrode, which stands facing the transparent electrodewith the liquid sample intervening therebetween, and

vi) means for applying a DC voltage across the opposite electrode andthe transparent electrode.

The first evanescent ellipsosensor in accordance with the presentinvention may be modified such that the sample analysis can also becarried out on the side of the opposite electrode which is provided forvoltage application. Specifically, the present invention still furtherprovides a second evanescent ellipsosensor, wherein the aforesaid firstevanescent ellipsosensor in accordance with the present invention ismodified such that it may further comprise:

a second prism, a second transparent electrode serving as the oppositeelectrode being formed on one surface of the second prism,

a second beam irradiating system for irradiating a second light beam,which has been polarized in a predetermined condition of polarization,from the side of the second prism such that the second light beam may betotally reflected from an interface between the liquid sample and thesecond transparent electrode, and

means for detecting a change in condition of polarization of the secondlight beam due to the total reflection.

The present invention also provides a first electrophoresis sensor,comprising:

i) an electrophoresis medium having been impregnated with a sample,

ii) a first electrode, which is constituted of a metal film and islocated such that it may be in contact with one end of theelectrophoresis medium,

iii) a second electrode, which is located on the side of the other endof the electrophoresis medium,

iv) means for applying a DC voltage across the first electrode and thesecond electrode, the DC voltage causing a substance, which is to bedetected and is contained in the sample, to migrate from the other endof the electrophoresis medium toward the one end thereof,

v) a prism, which located such that it may be in contact with the firstelectrode from the side opposite to the one end of the electrophoresismedium,

vi) a beam irradiating system for causing a light beam to pass throughthe prism and to impinge upon an interface between the prism and thefirst electrode such that various different angles of incidence may beobtained with respect to the interface, and

vii) a photo detecting means capable of detecting an intensity of thelight beam, which has been totally reflected from the interface, withrespect to each of the various different angles of incidence.

The present invention further provides a second electrophoresis sensor,comprising:

i) an electrophoresis medium having been impregnated with a sample,

ii) a first electrode, which is transparent and is located such that itmay be in contact with one end of the electrophoresis medium,

iii) a second electrode, which is located on the side of the other endof the electrophoresis medium,

iv) means for applying a DC voltage across the first electrode and thesecond electrode, the DC voltage causing a substance, which is to bedetected and is contained in the sample, to migrate from the other endof the electrophoresis medium toward the one end thereof,

v) a prism, which located such that it may be in contact with the firstelectrode from the side opposite to the one end of the electrophoresismedium,

vi) a beam irradiating system for irradiating a light beam, which hasbeen polarized in a predetermined condition of polarization, from theside of the prism such that the light beam may be totally reflected froman interface between the prism and the first electrode, and

vii) means for detecting a change in condition of polarization of thelight beam due to the total reflection.

A third electrophoresis sensor in accordance with the present inventionis designed for analyzing a sample containing a substance to bedetected, which has been labeled with a fluorescent substance.Specifically, the present invention still further provides a thirdelectrophoresis sensor, comprising:

i) an electrophoresis medium having been impregnated with a samplecontaining a substance, which is to be detected and has been labeledwith a fluorescent substance,

ii) a first electrode, which is transparent and is located such that itmay be in contact with one end of the electrophoresis medium,

iii) a second electrode, which is located on the side of the other endof the electrophoresis medium,

iv) means for applying a DC voltage across the first electrode and thesecond electrode, the DC voltage causing the substance, which is to bedetected and is contained in the sample, to migrate from the other endof the electrophoresis medium toward the one end thereof,

v) a prism, which located such that it may be in contact with the firstelectrode from the side opposite to the one end of the electrophoresismedium,

vi) a beam irradiating system for irradiating a light beam from the sideof the prism such that the light beam may be totally reflected from aninterface between the prism and the first electrode, and

vii) means for detecting fluorescence, which is produced by thefluorescent substance when the fluorescent substance is excited with anevanescent wave having leaked from the interface.

With the first surface plasmon sensor in accordance with the presentinvention, the DC voltage is applied across the metal film and theelectrode, which are located with the liquid sample interveningtherebetween. Therefore, the substance to be analyzed, which haselectric charges in the liquid sample, can be electro-deposited on themetal film. The polarity of the voltage may be selected in accordancewith whether the substance to be analyzed is a cation or an anion, orthe like.

Due to the electro-deposition, the concentration of the substance to beanalyzed becomes high in the portion of the liquid sample, which portionis in contact with the metal film. As a result, the total reflectioncancellation angle changes quickly and markedly. Therefore, thesubstance to be analyzed can be analyzed quickly and with a highsensitivity. In particular, in cases where a substance in a liquidsample is to be detected by the utilization of an antigen-antibodyreaction, i.e. in cases where, for example, an antigen (or an antibody)is fixed to the metal film and an antibody (or an antigen), which iscontained in the liquid sample and specifically adsorbs to the metalfilm, is to be detected, the antigen-antibody reaction is promoted bythe increase in concentration of the substance to be analyzed, andtherefore the effects described above can be obtained more markedly.

With the second surface plasmon sensor in accordance with the presentinvention, the electrode provided for voltage application is constitutedof the metal film, and the prism, the light source, the optical system,and the photo detecting means are located also for the electrodeprovided for voltage application, such that a sample analysis can alsobe carried out on the side of this electrode. Therefore, a substancehaving positive electric charges in the liquid sample and a substancehaving negative electric charges in the liquid sample can be analyzedsimultaneously.

With the third surface plasmon sensor in accordance with the presentinvention, the DC voltage is applied across the metal film and theelectrode, which are formed at positions spaced apart from each other onone surface of the prism. Therefore, as in the first and second surfaceplasmon sensors in accordance with the present invention, the substanceto be analyzed, which has electric charges in the liquid sample, can beelectro-deposited on the metal film. By virtue of the effects of theelectro-deposition, the substance to be analyzed, which is contained inthe liquid sample, can be analyzed quickly and with a high sensitivity.

With the fourth, fifth, and sixth surface plasmon sensors in accordancewith the present invention, the sensor film, to which an antigen or anantibody capable of undergoing an antigen-antibody reaction with asubstance to be analyzed, that is contained in the liquid sample, hasbeen fixed, is formed on the metal film. As a result, the substance tobe analyzed, that is contained in the liquid sample, combines with thesensor film. Therefore, the physical properties in the vicinity of thesensor means, which is constituted of the sensor film and the metalfilm, change markedly. Accordingly, the sensitivity of the sensor systemcan be kept high, and the rate of the antigen-antibody reaction can bekept high.

With the seventh surface plasmon sensor in accordance with the presentinvention, light coupling to the sensor means is carried out with thegrating for entry of the light beam and the grating for radiation of thelight beam, which are formed on the substrate. Therefore, multiplereflection interference, which will occur when a prism is utilized forthe coupling, can be prevented from occurring.

Also, with the seventh surface plasmon sensor in accordance with thepresent invention, wherein no prism is used and the light couplingsection is located in the plane-parallel form, the size of the surfaceplasmon sensor can be kept small, and adjustment of the optical axis canbe carried out easily.

With the first evanescent ellipsosensor in accordance with the presentinvention, the DC voltage is applied across the transparent electrodeand the opposite electrode, which are located with the liquid sampleintervening therebetween. Therefore, the substance to be analyzed, whichhas electric charges in the liquid sample, can be electro-deposited onthe transparent electrode. The polarity of the voltage may be selectedin accordance with whether the substance to be analyzed is a cation oran anion, or the like.

Due to the electro-deposition, the concentration of the substance to beanalyzed becomes high in the portion of the liquid sample, which portionis in contact with the transparent electrode. Therefore, the substanceto be analyzed can be analyzed with a high sensitivity. In particular,in cases where a substance in a liquid sample is to be detected by theutilization of an antigen-antibody reaction, i.e. in cases where, forexample, an antigen (or an antibody) is fixed to the transparentelectrode and an antibody (or an antigen), which is contained in theliquid sample and specifically adsorbs to the metal film, is to bedetected, the antigen-antibody reaction is promoted by the increase inconcentration of the substance, which is to be analyzed, in accordancewith the law of mass action, and therefore an analysis can be madequickly and with a high sensitivity.

Also, with the first evanescent ellipsosensor in accordance with thepresent invention, the electrode for voltage application, which isformed on one surface of the prism, is a transparent electrode.Therefore, as in the conventional apparatus wherein the electrode is notformed, the evanescent wave leaks to the liquid sample. Accordingly, thesample analysis can be carried out without being obstructed by theelectrode.

Such that the light beam maybe reflected totally, it is necessary forthe transparent electrode and the prism to be constituted of materialshaving refractive indexes higher than the refractive index of the liquidsample. Also, from the view point of preventing the reflection from theinterface between the prism and the transparent electrode and multiplereflection interference in the transparent electrode film, thetransparent electrode and the prism should preferably be constituted ofmaterials having an identical refractive index.

Particularly, with the second evanescent ellipsosensor in accordancewith the present invention, the opposite electrode is constituted of atransparent electrode, and the prism, the beam irradiating system, andthe means for detecting a change in condition of polarization arelocated also for the opposite electrode, such that a sample analysis canalso be carried out on the side of the opposite electrode. Therefore, asubstance having positive electric charges in the liquid sample and asubstance having negative electric charges in the liquid sample can beanalyzed simultaneously.

With the first electrophoresis sensor in accordance with the presentinvention, when a light beam impinges at an angle of incidence, which isnot smaller than the total reflection angle, upon the first electrodeconstituted of the metal film, an evanescent wave having an electricfield distribution occurs in the sample, which is in contact with thefirst electrode, and the surface plasmon is excited at the interfacebetween the metal film and the sample by the evanescent wave. When theangle of incidence becomes equal to a specific angle θ_(SP), the wavenumber of the evanescent wave and the wave number of the surface plasmonbecome equal to each other, and wave number matching is obtained. Inthis condition, energy of the light transfers to the surface plasmon. Asa result, the intensity of the light, which is totally reflected fromthe interface between the prism and the first electrode, becomesmarkedly low. This phenomenon occurs only when the incident light iscomposed of the P-polarized light component (normal to the metal film).

Therefore, the specific substance contained in the sample can bequantitatively analyzed by finding the angle of incidence θ_(SP), atwhich the intensity of the reflected light beam becomes low.

With the first electrophoresis sensor in accordance with the presentinvention, in this manner, only the substance having arrived at thefirst electrode is detected. A plurality of substances contained in thesample arrive at the first electrode after different lengths of time dueto a difference in migration speed. Therefore, the substances aretemporally separated from one another and detected.

As described above, with the first electrophoresis sensor in accordancewith the present invention, a plurality of substances contained in thesample are temporally separated from one another and detected.Therefore, exposure operations for recording the images of thesubstances by spatially separating them, development processes, and thelike, need not be carried out, and the substances contained in thesample can be analyzed easily.

Also, with the first electrophoresis sensor in accordance with thepresent invention, the substance contained in the sample is analyzed inaccordance with the intensity of the reflected light described above.Therefore, operations for labeling the substance in the sample need notbe carried out, and the analysis operation can be kept simple.

Effects of the second electrophoresis sensor in accordance with thepresent invention will be described hereinbelow. As described above,when a light beam traveling in a first medium is totally reflected by aninterface between the first medium and a second medium, which has arefractive index lower than the refractive index of the first medium,light referred to as the evanescent wave leaks to the second medium.When the polarized light beam impinges upon the interface, the conditionof polarization (i.e., the difference in phase between the P-polarizedlight component and the S-polarized light component) changes before thelight beam is totally reflected from the interface and after the lightbeam is totally reflected from the interface. The change in thecondition of polarization is inherent in accordance with the secondmedium which interacts with the evanescent wave.

With the second electrophoresis sensor in accordance with the presentinvention, the prism serves as the first medium, and the first electrodeand the substance having arrived at the first electrode serve as thesecond medium. Therefore, the substance in the sample can be analyzedquantitatively by detecting a change in condition of polarization of thelight beam due to the total reflection. In this manner, with the secondelectrophoresis sensor in accordance with the present invention, onlythe substance having arrived at the first electrode is detected. Also, aplurality of substances contained in the sample arrive at the firstelectrode after different lengths of time due to a difference inmigration speed. Therefore, the substances are temporally separated fromone another and detected.

Also, with the second electrophoresis sensor in accordance with thepresent invention, wherein the first electrode is a transparentelectrode, the evanescent wave leaks to the liquid sample, and thereforethe sample analysis can be made without being obstructed by the firstelectrode.

As described above, with the second electrophoresis sensor in accordancewith the present invention, a plurality of substances contained in thesample are temporally separated from one another and detected.Therefore, exposure operations for recording the images of thesubstances by spatially separating them, development processes, and thelike, need not be carried out, and the substances contained in thesample can be analyzed easily.

Further, with the second electrophoresis sensor in accordance with thepresent invention, the substance contained in the sample is analyzed bydetecting a change in condition of polarization. Therefore, operationsfor labeling the substance in the sample need not be carried out, andthe analysis operation can be kept simple.

Effects of the third electrophoresis sensor in accordance with thepresent invention will be described hereinbelow. With the thirdelectrophoresis sensor in accordance with the present invention, thelight beam is totally reflected from the interface between the prism andthe first electrode. At this time, an evanescent wave leaks from theinterface to the sample. The fluorescent substance serving as the labelis excited with the evanescent wave and produces the fluorescence. Thefluorescence thus produced is detected. Therefore, the substance havingarrived at the first electrode can be analyzed quantitatively inaccordance with the fluorescence.

In this manner, with the third electrophoresis sensor in accordance withthe present invention, only the substance having arrived at the firstelectrode is detected. Also, a plurality of substances contained in thesample arrive at the first electrode after different lengths of time dueto a difference in migration speed. Therefore, the substances aretemporally separated from one another and detected.

As described above, with the third electrophoresis sensor in accordancewith the present invention, a plurality of substances contained in thesample are temporally separated from one another and detected.Therefore, exposure operations for recording the images of thesubstances by spatially separating them, development processes, and thelike, need not be carried out, and the substances contained in thesample can be analyzed easily.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view showing a first embodiment of the surface plasmonsensor inaccordance with the present invention,

FIG. 2 is a graph showing approximate relationship between an angle ofincidence of a light beam upon a reflection interface and an intensityof a totally reflected light in a surface plasmon sensor,

FIG. 3 is a side view showing a second embodiment of the surface plasmonsensor in accordance with the present invention,

FIG. 4 is a side view showing a third embodiment of the surface plasmonsensor in accordance with the present invention,

FIG. 5 is a side view showing a fourth embodiment of the surface plasmonsensor in accordance with the present invention,

FIG. 6 is a plan view showing relationship between positions of a metalfilm and an electrode for voltage application in the fourth embodimentof the surface plasmon sensor shown in FIG. 5,

FIG. 7 is a plan view showing a different example of relationshipbetween positions of a metal film and an electrode for voltageapplication in the surface plasmon sensor in accordance with the presentinvention,

FIG. 8 is a plan view showing a further different example ofrelationship between positions of a metal film and an electrode forvoltage application in the surface plasmon sensor in accordance with thepresent invention,

FIG. 9 is a side view showing a fifth embodiment of the surface plasmonsensor in accordance with the present invention,

FIG. 10 is a side view showing a sixth embodiment of the surface plasmonsensor in accordance with the present invention,

FIG. 11 is a side view showing a first embodiment of the evanescentellipsosensor in accordance with the present invention,

FIG. 12 is a side view showing a second embodiment of the evanescentellipsosensor in accordance with the present invention,

FIG. 13 is a side view showing a first embodiment of the electrophoresissensor in accordance with the present invention,

FIG. 14 is a side view showing a second embodiment of theelectrophoresis sensor in accordance with the present invention,

FIG. 15 is a graph showing how a photo detection signal changes in thesecond embodiment of the electrophoresis sensor shown in FIG. 14,

FIG. 16 is a side view showing a third embodiment of the electrophoresissensor in accordance with the present invention,

FIG. 17 is a side view showing a fourth embodiment of theelectrophoresis sensor in accordance with the present invention, and

FIG. 18 is a side view showing a fifth embodiment of the electrophoresissensor in accordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will hereinbelow be described in further detailwith reference to the accompanying drawings.

FIG. 1 is a side view showing a first embodiment of the surface plasmonsensor in accordance with the present invention. As illustrated in FIG.1, the surface plasmon sensor comprises a prism 10 having a triangularcross-section, and a metal film 12 constituted of gold, silver, or thelike. The metal film 12 is formed on one surface (an upper surface inFIG. 1) of the prism 10 and is brought into contact with a liquid sample11. The surface plasmon sensor also comprises a light source 14, whichproduces a light beam 13 and maybe constituted of a semiconductor laser,or the like, and a cylindrical lens 15 for converging the light beam 13,which has been radiated in a divergent light condition from the lightsource 14, only in a plane normal to the major axis of the prism 10(i.e., a plane parallel to the plane of the sheet of FIG. 1). Thesurface plasmon sensor further comprises a photo detecting means 16 fordetecting an intensity of the light beam 13, which has been totallyreflected from an interface 10a between the prism 10 and the metal film12.

An electrode 17 is located at a position spaced an appropriate distancefrom the metal film 12 such that it may stand facing the metal film 12.The electrode 17 is supported by a tube-like support member 18 havingits center axis extending vertically in FIG. 1. The periphery of theregion sandwiched between the metal film 12 and the electrode 17 isclosed by the support member 18. The metal film 12 and the electrode 17are respectively connected to a positive pole and a negative pole of aDC power source 19.

The light beam 13 is converged in the manner described above by theaction of the cylindrical lens 15. Therefore, as exemplified by theminimum angle of incidence θ₁ and the maximum angle of incidence θ₂ inFIG. 1, the light beam 13 contains components impinging at variousdifferent angles of incidence θ upon the interface 10a. The angles ofincidence θ are set to be not smaller than the total reflection angle.As a result, the light beam 13 is .totally reflected from the interface10a, and the reflected light beam 13 contains the components, which havebeen reflected at various different angles of reflection.

As the photo detecting means 16, means having a light receiving section,which extends in the direction that is capable of receiving all of thecomponents of the light beam 13 having been reflected at differentangles of reflection in the manner described above, is employed. Thephoto detecting means 16 may be constituted of a charge coupled device(CCD) line sensor, or the like. A photo detection signal S is obtainedfrom each of light receiving elements of the photo detecting means 16.Therefore, the photo detection signal S represents the intensity of thelight beam 13 with respect to each of the various different angles ofreflection (i.e., with respect to each of the various different anglesof incidence).

How a sample analysis is carried out in the surface plasmon sensorhaving the constitution described above will be described hereinbelow.The region between the metal film 12 and the electrode 17 is filled withthe liquid sample 11, which contains an anionic substance to be analyzed30. Also, the DC power source 19 applies a DC voltage across the metalfilm 12 and the electrode 17. The light beam 13 is converged by theaction of the cylindrical lens 15 in the manner described above and isirradiated toward the metal film 12. The light beam 13 is totallyreflected from the interface 10a between the metal film 12 and the prism10, and the reflected light beam 13 is detected by the photo detectingmeans 16.

As described above, the photo detection signal S obtained from each ofthe light receiving elements of the photo detecting means 16 representsthe intensity I of the totally reflected light beam 13 with respect toeach of the angles of incidence θ. FIG. 2 approximately shows therelationship between the intensity I of the reflected light and theangles of incidence θ.

The light impinging at a specific angle of incidence θ_(SP) upon theinterface 10a excites surface plasmon at an interface between the metalfilm 12 and the liquid sample 11. As for the light impinging at thespecific angle of incidence θ_(SP) upon the interface 10a, the intensityI of the reflected light becomes markedly low. From the photo detectionsignal S obtained from each of the light receiving elements of the photodetecting means 16, the specific angle of incidence θ_(SP) can bedetermined. As described above in detail, the substance to be analyzed30 contained in the liquid sample 11 can be analyzed quantitatively inaccordance with the value of θ_(SP).

Also, since the DC voltage is applied across the metal film 12 and theelectrode 17, which are connected to the DC power source 19, the anionicsubstance to be analyzed 30, which is contained in the liquid sample 11,is electro-deposited on the metal film 12. Therefore, the concentrationof the substance to be analyzed 30 becomes high at the portion of theliquid sample 11, which portion is in contact with the metal film 12,and the substance to be analyzed 30 can be analyzed quickly and with ahigh sensitivity. As the anionic substance to be analyzed 30, an antigen(α-FP) dispersed in PBS, or the like, may be mentioned.

In cases where the substance to be analyzed 30, which is contained inthe liquid sample 11, is detected by the utilization of anantigen-antibody reaction, it is often desired that the relationshipbetween the intensity I of the reflected light and the angles ofincidence θ can be detected on the real time basis as time passes whilethe antigen-antibody reaction is proceeding. With the first embodimentof the surface plasmon sensor in accordance with the present invention,wherein the substance to be analyzed 30 can be detected quickly asdescribed above, the real time detection can be completed quickly.

In the first embodiment of the surface plasmon sensor in accordance withthe present invention, in order for the various different angles ofincidence θ to be obtained, the light beam 13 having a comparativelylarge beam diameter is irradiated such that it may be converged on theinterface 10a. Alternatively, various different angles of incidence θmay be obtained by deflecting a light beam having a comparatively smallbeam diameter.

A second embodiment of the surface plasmon sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.3. In FIG. 3 and in FIGS. 4 to 8, similar elements are numbered with thesame reference numerals with respect to FIG. 1.

The second embodiment of the surface plasmon sensor shown in FIG. 3 isbasically similar to the first embodiment of FIG. 1, except that thesecond embodiment further comprises a second prism 20, a second lightsource 24 for producing a light beam 23, a second cylindrical lens 25,and a second photo detecting means 26. In this case, as the electrode17, a film of a metal, such as gold or silver, is employed.

The elements newly added to the second embodiment of the surface plasmonsensor in accordance with the present invention constitute anothersurface plasmon sensor by utilizing the electrode 17, which isconstituted of the metal film, in the same manner as that of the metalfilm 12. Specifically, the second prism 20, the second light source 24,the second cylindrical lens 25, and the second photo detecting means 26respectively have the same actions as those of the prism 10, the lightsource 14, the cylindrical lens 15, and the photo detecting means 16.

In the second embodiment of FIG. 3, the analysis of the anionicsubstance to be analyzed 30, which is contained in the liquid sample 11,is carried out in the same manner as that described above. Also, in thesecond embodiment of FIG. 3, a cationic substance to be analyzed 31,which is contained in the liquid sample 11, is electro-deposited on theelectrode 17. Therefore, with the second prism 20, the second lightsource 24, the second cylindrical lens 25, and the second photodetecting means 26, an analysis of the substance to be analyzed 31 canbe carried out in the same manner.

In such cases, the concentration of the substance to be analyzed 31becomes high at the portion of the liquid sample 11, which portion is incontact with the electrode 17, and the substance to be analyzed 31 canbe analyzed quickly and with a high sensitivity.

A third embodiment of the surface plasmon sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.4. The third embodiment of the surface plasmon sensor shown in FIG. 4 isbasically similar to the first embodiment of FIG. 1, except that asensor film 40 is formed on the metal film 12, and the liquid sample 11is brought into direct contact with the sensor film 40. An antigen or anantibody capable of undergoing an antigen-antibody reaction with thesubstance to be analyzed 30, which is contained in the liquid sample 11,has been fixed to the sensor film 40. By way of example, in cases wherethe substance to be analyzed 30 is the aforesaid antigen (α-FP), IgG, orthe like, may be employed as the antibody.

In the third embodiment of FIG. 4, the substance to be analyzed 30,which is contained in the liquid sample 11, is attracted toward themetal film 12 by the aforesaid electro-deposition effects and combineswith the sensor film 40 by the antigen-antibody reaction. Therefore, thephysical properties change markedly in the vicinity of the sensorsection, which is constituted of the sensor film 40 and the metal film12. Accordingly, the sensitivity of the sensor system can be kept high,and the rate of the antigen-antibody reaction can be kept high.

A fourth embodiment of the surface plasmon sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.5. The fourth embodiment of the surface plasmon sensor shown in FIG. 5is basically similar to the first embodiment of FIG. 1, except that,instead of the metal film 12 and the electrode 17 facing each other, themetal film 12 and the electrode 17 are located at positions spaced apartfrom each other on one surface of the prism 10. FIG. 6 is a plan viewshowing the relationship between the positions of the metal film 12 andthe electrode 17 in the fourth embodiment of FIG. 5.

In the fourth embodiment of FIG. 5, when the DC power source 19 appliesthe DC voltage across the metal film 12 and the electrode 17, theanionic substance to be analyzed 30, which is contained in the liquidsample 11, is attracted toward the metal film 12 by the electrodeposition effects. Therefore, the concentration of the substance to beanalyzed 30 becomes high at the portion of the liquid sample 11, whichportion is close to the metal film 12, and the substance to be analyzed30 can be analyzed quickly and with a high sensitivity.

In the fourth embodiment of FIG. 5, a sensor film 40, which is of thesame type as that employed in the third embodiment of FIG. 4, is formedon the metal film 12. Therefore, in this embodiment, the physicalproperties change markedly in the vicinity of the sensor section, whichis constituted of the sensor film 40 and the metal film 12. Accordingly,the sensitivity of the sensor system can be kept high, and the rate ofthe antigen-antibody reaction can be kept high.

In cases where the metal film 12 and the electrode 17 are formed atpositions spaced apart from each other on one surface of the prism 10 asin the fourth embodiment of FIG. 5, the shapes of the metal film 12 andthe electrode 17 are not limited to those shown in FIG. 6 and may bedesigned as illustrated in, for example, FIGS. 7 and 8.

In cases where the shapes illustrated in FIG. 8 are employed, theso-called multi-channel constitution can be obtained. Specifically, insuch cases, the substance to be analyzed 30, which is contained in theliquid sample 11, is attracted to each of the three comb teeth-likeportions of the metal film 12. Therefore, three sensor sections can beobtained, and the analysis can be carried out simultaneously at thethree sensor sections.

FIG. 9 is a side view showing a fifth embodiment of the surface plasmonsensor in accordance with the present invention. As illustrated in FIG.9, the surface plasmon sensor comprises a light source 101, whichproduces a light beam 113 and may be constituted of a semiconductorlaser, or the like, and a glass substrate 105. The surface plasmonsensor also comprises a grating 106 for entry of the light beam, whichis formed at a portion of one surface (the lower surface in FIG. 9) ofthe glass substrate 105, and a sensor means (a metal film) 110, which isformed on the other surface of the glass substrate 105 and is broughtinto contact with a sample 111. The grating 106 for entry of the lightbeam diffracts the light beam 113 and causes it to enter into the glasssubstrate 105. The sensor means 110 may be constituted of gold, silver,or the like. The surface plasmon sensor further comprises a grating 107for radiation of the light beam, which is formed at a portion of the onesurface (the lower surface in FIG. 9) of the glass substrate 105. Thegrating 107 for radiation of the light beam diffracts the light beam 113having been totally reflected from an interface 110a between the glasssubstrate 105 and the metal film 110 and radiates the light beam 113 outof the glass substrate 105. The surface plasmon sensor still furthercomprises a photo detecting means 115 for detecting the light beam 113,which has been radiated out of the glass substrate 105.

The light source 101 is rotated by a goniometer (not shown), such thatthe angles of incidence of the light beam 113 upon the glass substrate105 and upon the metal film 110 may take various values. Specifically,in the fifth embodiment of FIG. 9, the goniometer corresponds to theoptical system for causing the light beam to impinge upon the substratein the aforesaid seventh surface plasmon sensor in accordance with thepresent invention. As exemplified by the minimum angle of incidence θ₁and the maximum angle of incidence θ₂ in FIG. 9, the light beam 113impinges at various different angles of incidence θ upon the interface110a between the glass substrate 105 and the metal film 110. The anglesof incidence θ are set to be not smaller than the critical angle oftotal reflection, such that the light beam 113 may be totally reflectedfrom the interface 110a.

The angle of reflection of the light beam 113, which is reflected fromthe interface 110a, changes in accordance with the change in angle ofincidence. Therefore, as the photo detecting means 115, means comprisinglight receiving elements arrayed in the direction, along which the angleof reflection changes, is employed. By way of example, the photodetecting means 115 may be constituted of a CCD line sensor.

In lieu of the goniometer being used, various different angles ofincidence may be obtained by deflecting the light beam with agalvanometer mirror, which is described in, for example, Japanese PatentApplication No. 8(1996)-109367. Also, the photo detecting means 115 maybe rotated by a goniometer in accordance with the change in angle ofreflection due to the rotation of the light source, and the intensity ofthe light beam may thereby be detected.

How a sample analysis is carried out in the fifth embodiment of thesurface plasmon sensor shown in FIG. 9 will be described hereinbelow.The sample 11 subjected to the analysis is located such that it may incontact with the metal film 110. The light beam 113 is set to becomposed of P-polarized light component and is caused by the goniometerto enter at various different angles of incidence from the grating 106for entry of the light beam into the glass substrate 105. The light beam113 is diffracted by the grating 106 for entry of the light beam and iscaused to impinge at angles of incidence θ upon the metal film 110. Thelight beam 113 is then totally reflected from the interface 110a betweenthe metal film 110 and the glass substrate 105, diffracted by thegrating 107 for radiation of the light beam, and radiated out of theglass substrate 105. The light beam 113 having thus been radiated out ofthe glass substrate 105 is detected by the photo detecting means 115.

A photo detection signal S obtained from each of the light receivingelements of the photo detecting means 115 represents the intensity I ofthe totally reflected light beam 113 with respect to each of the anglesof incidence θ upon the interface 110a between the glass substrate 105and the metal film 110. FIG. 2 approximately shows the relationshipbetween the intensity I of the reflected light and the angles ofincidence θ.

The light impinging at a specific angle of incidence (a total reflectioncancellation angle) θ_(SP) upon the interface 110a excites surfaceplasmon at an interface between the metal film 110 and the sample 111.As for the light impinging at the specific angle of incidence θ_(SP)upon the interface 110a, the intensity I of the reflected light becomesmarkedly low. From the photo detection signal S obtained from each ofthe light receiving elements of the photo detecting means 115, the totalreflection cancellation angle θ_(SP) can be determined. As describedabove in detail, a substance to be analyzed contained in the sample 111can be analyzed quantitatively in accordance with the value of the totalreflection cancellation angle θ_(SP).

As an alternative to the constitution of the fifth embodiment of FIG. 9,as illustrated in FIG. 10, a collimator lens 102 and a converging lens103 may be employed as the optical system, and the converged light beammay be irradiated to the interface 110a. As exemplified by the minimumangle of incidence θ₁ and the maximum angle of incidence θ₂ in FIG. 10,the converged light beam contains components impinging at variousdifferent angles of incidence θ upon the interface 110a. Therefore, thelight beam, which has been totally reflected from the interface 110a,contains the components, which have been reflected at various differentangles of reflection. The intensity of the light beam having beenradiated out of the glass substrate 105 is detected by the photodetecting means 115. As the photo detecting means 115, a CCD linesensor, a photodiode, a two-part photodiode as described in, forexample, Japanese Patent Application No. 8(1996)-109366, a photodiodearray, or the like, may be employed. As in the fourth embodiment of FIG.9, the total reflection cancellation angle θ_(SP) can be found from thephoto detection signal, and a specific substance contained in the sample111 can be analyzed quantitatively.

In the embodiments described above, the total reflection cancellationangle θ_(SP) is obtained from the intensity of the reflected light withrespect to various different angles of incidence. Alternatively, thetotal reflection cancellation angle θ_(SP) may be obtained by utilizingthe characteristics such that the intensity of the reflected light withrespect to a certain angle of incidence changes in accordance with thevalue of the total reflection cancellation angle θ_(SP). For example,the angle of incidence of the light beam may be set at a predeterminedangle smaller than the total reflection cancellation angle θ_(SP), andthe total reflection cancellation angle θ_(SP) may be obtained inaccordance with the intensity of the reflected light, which is obtainedat this time.

Embodiments of the evanescent ellipsosensor in accordance with thepresent invention will be described hereinbelow.

FIG. 11 is a side view showing a first embodiment of the evanescentellipsosensor in accordance with the present invention. As illustratedin FIG. 11, the evanescent ellipsosensor comprises a prism 210 having atriangular cross-section, a transparent electrode 212, which is formedon one surface (an upper surface in FIG. 11) of the prism 210 and isbrought into contact with a liquid sample 211, and a light source 214,which produces a light beam 213 and may be constituted of a laser. Theevanescent ellipsosensor also comprises a polarizer 215 and aquarter-wave plate 216 for controlling the condition of polarization ofthe light beam 213, which has been produced by the light source 214. Theevanescent ellipsosensor further comprises an analyzer 217, which islocated in the optical path of the light beam 213 having been totallyreflected from an interface 210a between the prism 210 and thetransparent electrode 212, and a photo detecting means 218 for detectingthe intensity of the light beam 213 having passed through the analyzer217.

An opposite electrode 219 is located at a position spaced an appropriatedistance from the transparent electrode 212 such that it may standfacing the transparent electrode 212. The opposite electrode 219 issupported by a tube-like support member 220 having its center axisextending vertically in FIG. 11. The periphery of the region sandwichedbetween the transparent electrode 212 and the opposite electrode 219 isclosed by the support member 220. The transparent electrode 212 and theopposite electrode 219 are respectively connected to a positive pole anda negative pole of a DC power source 221.

The beam irradiating system, which is composed of the light source 214,the polarizer 215, and the quarter-wave plate 216, is located such thatthe light beam 213 may impinge at an angle of incidence not smaller thanthe total reflection angle upon the interface 210a. Also, the polarizer215 and the quarter-wave plate 216 convert the light beam 213 into anelliptically polarized light such that it may constitute a linearlypolarized light after being totally reflected from the interface 210a.The analyzer 217 is rotated around the optical axis.

How a sample analysis is carried out in the evanescent ellipsosensorhaving the constitution described above will be described hereinbelow.The region between the transparent electrode 212 and the oppositeelectrode 219 is filled with the liquid sample 211, which contains ananionic substance to be analyzed 230. Also, the DC power source 221applies a DC voltage across the transparent electrode 212 and theopposite electrode 219. The light beam 213 having been converted intothe elliptically polarized light is irradiated toward the transparentelectrode 212. The light beam 213 is totally reflected from theinterface 210a between the transparent electrode 212 and the prism 210,and the reflected light beam 213 is detected by the photo detectingmeans 218.

When the light beam 213 is totally reflected from the interface 210a, adifference in phase between the P-polarized light component (i.e., thepolarized light component having a plane of vibration parallel to theinterface 210a) and the S-polarized light component (i.e., the polarizedlight component having a plane of vibration normal to the interface210a) varies for the incident light and the reflected light. Asdescribed above, the change in difference in phase, i.e. the change incondition of polarization, due to the total reflection reflects thephysical properties and the total amount of the substance to be analyzed230, which adheres to the transparent electrode 212. Therefore, theanalyzer 217 is rotated such that an output S of the photo detectingmeans 218 may become smallest. At this time, from the rotation angle ofthe analyzer 217, the change in condition of polarization due to thetotal reflection, and consequently the physical properties and the totalamount of the substance to be analyzed 230, can be determined.

Also, since the DC voltage is applied across the transparent electrode212 and the opposite electrode 219, which are connected to the DC powersource 221, the anionic substance to be analyzed 230, which is containedin the liquid sample 211, is electro-deposited on the transparentelectrode 212. Therefore, the concentration of the substance to beanalyzed 230 becomes high at the portion of the liquid sample 211, whichportion is in contact with the transparent electrode 212, and thesubstance to be analyzed 230 can be analyzed quickly and with a highsensitivity. As the anionic substance to be analyzed 230, human serumtransferring dissolved in sodium hydroxide, or the like, may bementioned.

In cases where the substance to be analyzed 230, which is contained inthe liquid sample 211, is detected by the utilization of anantigen-antibody reaction, it is often desired that the change incondition of polarization of the light beam 213 can be detected on thereal time basis as time passes while the antigen-antibody reaction isproceeding. With the first embodiment of the evanescent ellipsosensor inaccordance with the present invention, wherein the substance to beanalyzed 230 can be detected quickly as described above, the real timedetection can be completed quickly.

In the first embodiment of the evanescent ellipsosensor in accordancewith the present invention, the change in condition of polarization ofthe light beam 213 due to the total reflection is detected with theanalyzer 217, which rotates, and the photo detecting means 218.Alternatively, the change in condition of polarization of the light beam213 may be detected with one of other known techniques, for example, atechnique for utilizing a photoelastic modulator (PEM).

A second embodiment of the evanescent ellipsosensor in accordance withthe present invention will be described hereinbelow with reference toFIG. 12. In FIG. 12, similar elements are numbered with the samereference numerals with respect to FIG. 11.

The second embodiment of the evanescent ellipsosensor shown in FIG. 12is basically similar to the first embodiment of FIG. 11, except that thesecond embodiment further comprises a second prism 240, a second lightsource 244 for producing a second light beam 243, a second polarizer245, a second quarter-wave plate 246, a second analyzer 247, and asecond photo detecting means 248. In this case, as the oppositeelectrode 219, a transparent electrode is employed.

The elements newly added to the second embodiment of the evanescentellipsosensor in accordance with the present invention constituteanother evanescent ellipsosensor by utilizing the opposite electrode219, which is constituted of the transparent electrode, in the samemanner as that of the transparent electrode 212. Specifically, thesecond prism 240, the second light source 244, the second polarizer 245,the second quarter-wave plate 246, the second analyzer 247, and thesecond photo detecting means 248 respectively have the same actions asthose of the prism 210, the light source 214, the polarizer 215, thequarter-wave plate 216, the analyzer 217, and the photo detecting means218.

In the second embodiment of the evanescent ellipsosensor shown in FIG.12, the analysis of the anionic substance to be analyzed 230, which iscontained in the liquid sample 211, is carried out in the same manner asthat described above. Also, in the second embodiment of FIG. 12, acationic substance to be analyzed 231, which is contained in the liquidsample 211, is electro-deposited on the opposite electrode 219.Therefore, with the second prism 240, the second light source 244, thesecond polarizer 245, the second quarter-wave plate 246, the secondanalyzer 247, and the second photo detecting means 248, an analysis ofthe substance to be analyzed 231 can be carried out in the same manner.

In such cases, the concentration of the substance to be analyzed 231becomes high at the portion of the liquid sample 211, which portion isin contact with the opposite electrode 219, and the substance to beanalyzed 231 can be analyzed quickly and with a high sensitivity.

Embodiments of the electrophoresis sensor in accordance with the presentinvention will be described hereinbelow.

FIG. 13 is a side view showing a first embodiment of the electrophoresissensor in accordance with the present invention. As illustrated in FIG.13, the electrophoresis sensor comprises a heat-insulating vessel 310filled with a liquid sample 311, a first electrode 312 which is locatedat the bottom of the heat-insulating vessel 310 such that it may be incontact with the liquid sample 311, and a second electrode 313 which islocated at the top of the heat-insulating vessel 310 such that it may bein contact with the liquid sample 311. The electrophoresis sensor alsocomprises a DC power source 314 for applying a DC voltage across thefirst electrode 312 and the second electrode 313. As the first electrode312, by way of example, a metal film constituted of gold, silver, or thelike, is employed.

The electrophoresis sensor further comprises a prism 315, which has atriangular prism-like shape and is brought into contact with the firstelectrode 312 from below (i.e., from the exterior of the heat-insulatingvessel 310. The electrophoresis sensor still further comprises a lightsource 317, which produces a light beam 316 and may be constituted of asemiconductor laser, or the like, and a cylindrical lens 318 forconverging the light beam 316, which has been radiated in a divergentlight condition from the light source 317, only in a plane normal to themajor axis of the prism 315 (i.e., a plane parallel to the plane of thesheet of FIG. 13). The electrophoresis sensor also comprises a photodetecting means 319 for detecting an intensity of the light beam 316,which has been totally reflected from an interface 315a between theprism 315 and the first electrode 312.

The light beam 316 is converged in the manner described above by theaction of the cylindrical lens 318. Therefore, as exemplified by theminimum angle of incidence θ₁ and the maximum angle of incidence θ₂ inFIG. 13, the light beam 316 contains components impinging at variousdifferent angles of incidence θ upon the interface 315a. The angles ofincidence θ are set to be not smaller than the total reflection angle.As a result, the light beam 316 is totally reflected from the interface315a, and the reflected light beam 316 contains the components, whichhave been reflected at various different angles of reflection.

As the photo detecting means 319, means having a light receivingsection, which extends in the direction that is capable of receiving allof the components of the light beam 316 having been reflected atdifferent angles of reflection in the manner described above, isemployed. The photo detecting means 319 may be constituted of a CCD linesensor, or the like. A photo detection signal S1 is obtained from eachof light receiving elements of the photo detecting means 319. Therefore,the photo detection signal S1 represents the intensity of the light beam316 with respect to each of the various different angles of reflection(i.e., with respect to each of the various different angles ofincidence).

How a sample analysis is carried out in the first embodiment of theelectrophoresis sensor having the constitution described above will bedescribed hereinbelow. A gel sheet 320, which is constituted of apolyacrylamide gel, or the like, and serves as an electrophoresismedium, is located in the heat-insulating vessel 310 such that one end(the lower end in FIG. 13) of the gel sheet 320 may be in contact withthe first electrode 312. Also, the heat-insulating vessel 310 is filledwith the liquid sample 311. The DC power source 314 applies the DCvoltage across the first electrode 312 and the second electrode 313. Thelight beam 316 is converged by the action of the cylindrical lens 318 inthe manner described above and is irradiated toward the first electrode312. The light beam 316 is totally reflected from the interface 315abetween the first electrode 312 and the prism 315, and the reflectedlight beam 316 is detected by the photo detecting means 319.

As described above, the photo detection signal S1 obtained from each ofthe light receiving elements of the photo detecting means 319 representsthe intensity I of the totally reflected light beam 316 with respect toeach of the angles of incidence θ. FIG. 2 approximately shows therelationship between the intensity I of the reflected light and theangles of incidence θ.

The light impinging at a specific angle of incidence θ_(SP) upon theinterface 315a excites surface plasmon at an interface between the firstelectrode 312 and the liquid sample 311. As for the light impinging atthe specific angle of incidence θ_(SP) upon the interface 315a, theintensity I of the reflected light becomes markedly low. From the photodetection signal S1 obtained from each of the light receiving elementsof the photo detecting means 319, the specific angle of incidence θ_(SP)can be determined. As described above in detail, a substance to beanalyzed, which is contained in the liquid sample 311, can be analyzedquantitatively in accordance with the value of θ_(SP).

Also, since the DC voltage is applied across the first electrode 312 andthe second electrode 313, which are connected to the DC power source314, a plurality of substances contained in the liquid sample 311migrate through the gel sheet 320 and arrive one after another at thefirst electrode 312. At this time, the plurality of the substancesarrive at the first electrode 312 at different time intervals due todifferences in migration speed. Specifically, the substance, which isquantitatively analyzed in accordance with the photo detection signalS1, is each of the substances having thus migrated through the gel sheet320. The substances are detected separately from one another inaccordance with the photo detection signal S1, the value of whichchanges with the passage of time.

In the first embodiment of the electrophoresis sensor in accordance withthe present invention, in order for the various different angles ofincidence θ to be obtained, the light beam 316 having a comparativelylarge beam diameter is irradiated such that it may be converged on theinterface 315a. Alternatively, various different angles of incidence θmay be obtained by deflecting a light beam having a comparatively smallbeam diameter.

A second embodiment of the electrophoresis sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.14. In FIG. 14 and in FIGS. 16, 17, and 18, similar elements arenumbered with the same reference numerals with respect to FIG. 13.

In the second embodiment of the electrophoresis sensor shown in FIG. 14,the constitution for electrophoresis is basically similar to that in thefirst embodiment of FIG. 13, and only the sensor section for light isdifferent from that in the first embodiment. Specifically, theelectrophoresis sensor shown in FIG. 14 comprises the prism 315 which isof the same type as that in the first embodiment shown in FIG. 13, atransparent first electrode 330, which is formed on one surface (anupper surface in FIG. 14) of the prism 315 and is brought into contactwith the liquid sample 311, and a light source 332, which produces alight beam 331 and may be constituted of a laser. The electrophoresissensor also comprises a polarizer 333 and a quarter-wave plate 334 forcontrolling the condition of polarization of the light beam 331, whichhas been produced by the light source 332. The electrophoresis sensorfurther comprises an analyzer 335, which is located in the optical pathof the light beam 331 having been totally reflected from the interface315a between the prism 315 and the transparent first electrode 330, anda photo detecting means 336 for detecting the intensity of the lightbeam 331 having passed through the analyzer 335.

The beam irradiating system, which is composed of the light source 332,the polarizer 333, and the quarter-wave plate 334, is located such thatthe light beam 331 may impinge at an angle of incidence not smaller thanthe total reflection angle upon the interface 315a. Also, the polarizer333 and the quarter-wave plate 334 convert the light beam 331 into acircularly polarized light immediately before the light beam 331impinges upon the interface 315a. The analyzer 335 is rotated around theoptical axis.

How a sample analysis is carried out in the second embodiment of theelectrophoresis sensor having the constitution described above will bedescribed hereinbelow. The aforesaid gel sheet 320 is located in theheat-insulating vessel 310 such that one end (the lower end in FIG. 14)of the gel sheet 320 may be in contact with the first electrode 330.Also, the heat-insulating vessel 310 is filled with the liquid sample311. The DC power source 314 applies the DC voltage across the firstelectrode 330 and the second electrode 313. The light beam 331, whichhas been converted into the circularly polarized light, is irradiatedtoward the first electrode 330. The light beam 331 is totally reflectedfrom the interface 315a between the first electrode 330 and the prism315, and the reflected light beam 331 is detected by the photo detectingmeans 336.

When the light beam 331 is totally reflected from the interface 315a, adifference in phase between the P-polarized light component (i.e., thepolarized light component having a plane of vibration parallel to theinterface 315a) and the S-polarized light component (i.e., the polarizedlight component having a plane of vibration normal to the interface315a) varies for the incident light and the reflected light. The changein difference in phase, i.e. the change in condition of polarization,due to the total reflection reflects the physical properties and thetotal amount of the substance to be analyzed, which adheres to the firstelectrode 330. Therefore, ellipticity of the polarized light isdetermined from an output S2 of the photo detecting means 336, and ashift from the circular polarization is thus investigated. In thismanner, the change in condition of polarization due to the totalreflection, and consequently the physical properties and the totalamount of the substance to be analyzed, can be determined.

Also, since the DC voltage is applied across the first electrode 330 andthe second electrode 313, the plurality of the substances contained inthe liquid sample 311 migrate through the gel sheet 320 and arrive oneafter another at the first electrode 330. At this time, the plurality ofthe substances arrive at the first electrode 330 at different timeintervals due to differences in migration speed. Therefore, thesubstances can be detected temporally separately from one another.

In the second embodiment of the electrophoresis sensor in accordancewith the present invention, the change in condition of polarization ofthe light beam 331 due to the total reflection is detected with theanalyzer 335, which rotates, and the photo detecting means 336.Alternatively, the change in condition of polarization of the light beam331 may be detected with one of other known techniques. For example, incases where the analyzer 335 is kept stationary without being rotated,the photo detection signal S2 changes moment by moment in accordancewith the change in condition of polarization. Therefore, the change incondition of polarization can be detected in the real time basis inaccordance with the value of the photo detection signal S2. FIG. 15shows how the photo detection signal S2 changes with the passage oftime.

One example of the separative detection of substances is the separativedetection of collagen polypeptide chains. Molecular weights of anα-chain, a β-chain, and a γ-chain in collagen are respectively 96,000,192,000, and 288,000. When a voltage is applied for the detection ofthese substances such that the first electrode 330 may serve as apositive electrode, the respective substances migrate toward the firstelectrode 330. A substance having a small molecular weight migrates at ahigh migration speed. Therefore, α-chain, the β-chain, and the γ-chainarrive in this order at the first electrode 330. When each of thesesubstances arrives at the first electrode 330, the dielectric constant(the refractive index) in the vicinity of the surface of the firstelectrode 330 changes in accordance with each substance, and theaforesaid condition of polarization is thereby caused to change. Asdescribed above, the change in condition of polarization is detected asa change in photo detection signal S2.

A third embodiment of the electrophoresis sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.16. In the third embodiment of the electrophoresis sensor shown in FIG.16, the constitution for electrophoresis is basically similar to that inthe first embodiment of FIG. 13, and only the sensor section for lightis different from that in the first embodiment. Specifically, theelectrophoresis sensor shown in FIG. 16 comprises a prism 340, which hasan approximately trapezoidal cross-section and is located such that itmay be in contact with the transparent first electrode 330, and a lightsource 342, which irradiates a light beam 341 to the prism 340 and maybe constituted of a laser. The electrophoresis sensor also comprises aconverging lens 343 for converging fluorescence 346, which will bedescribed later, and a wavelength filter 344 for selectivelytransmitting the fluorescence 346, and a photodetector 345 for detectingthe fluorescence 346. The light source 342 is located such that thelight beam 341 having been produced by it may be totally reflected froman interface 340a between the prism 340 and the first electrode 330.

How a sample analysis is carried out in the third embodiment of theelectrophoresis sensor having the constitution described above will bedescribed hereinbelow. The aforesaid gel sheet 320 is located in theheat-insulating vessel 310 such that one end (the lower end in FIG. 16)of the gel sheet 320 may be in contact with the first electrode 330.Also, the heat-insulating vessel 310 is filled with the liquid sample311. A substance to be detected, which is contained in the liquid sample311, is labeled with a coloring matter which is one kind of fluorescentsubstances. The DC power source 314 applies the DC voltage across thefirst electrode 330 and the second electrode 313. Also, the light beam341 is irradiated toward the first electrode 330.

When the light beam 341 is totally reflected from the interface 340a, anevanescent wave leaks from the interface 340a toward the first electrode330. At this time, if the substance, which is labeled with the coloringmatter and is contained in the liquid sample 311, has arrived at thefirst electrode 330, the coloring matter will be excited with theevanescent wave and will generate the fluorescence 346. The fluorescence346 is converged by the converging lens 343, guided to the photodetector345, and detected by the photodetector 345. Thus in this embodiment, thesubstance, which is labeled with the coloring matter and is contained inthe liquid sample 311, can be detected in accordance with a photodetection signal S3 obtained from the photodetector 345.

In the third embodiment of the electrophoresis sensor, since the DCvoltage is applied across the first electrode 330 and the secondelectrode 313, the plurality of the substances contained in the liquidsample 311 migrate through the gel sheet 320 and arrive one afteranother at the first electrode 330. At this time, the plurality of thesubstances arrive at the first electrode 330 at different time intervalsdue to differences in migration speed. Therefore, the substances can bedetected temp orally separately from one another.

A fourth embodiment of the electrophoresis sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.17. In the fourth embodiment of the electrophoresis sensor shown in FIG.17, the sensor section for light, which is shown in FIG. 13, is added tothe constitution shown in FIG. 16.

When a plurality of substances, which are contained in the liquid sample311, are to be detected separately from one another with theelectrophoresis sensor of FIG. 17, only a specific substance is labeledwith a scoring matter. All of the plurality of the substances aredetected with the sensor section utilizing the surface plasmon (the samesensor section as that shown in FIG. 13). Of the substances, thesubstance labeled with the coloring matter can also be detected withfluorescence observation. Therefore, in cases where a known specificsubstance is labeled with the coloring matter and is utilized as areference sample, a reference signal can be obtained from thefluorescence detection signal S3, and the other substances can beanalyzed in accordance with the photo detection signal S1.

A fifth embodiment of the electrophoresis sensor in accordance with thepresent invention will be described hereinbelow with reference to FIG.18. In the fifth embodiment of the electrophoresis sensor shown in FIG.18, the sensor section for light, which is shown in FIG. 14, is added tothe constitution shown in FIG. 16.

When a plurality of substances, which are contained in the liquid sample311, are to be detected separately from one another with theelectrophoresis sensor of FIG. 18, a known specific substance is labeledwith a coloring matter and is utilized as a reference sample. Therefore,as in the electrophoresis sensor of FIG. 17, a reference signal can beobtained from the fluorescence detection signal S3, and the othersubstances can be analyzed in accordance with the photo detection signalS2.

What is claimed is:
 1. A surface plasmon sensor, comprising:i) a prism,ii) a metal film formed on one surface of the prism and contacting aliquid sample, iii) a light source for producing a light beam, iv) anoptical system form causing the light beam to pass through the prism andto impinge upon an interface between the prism and the metal film suchthat various different angles of incidence may be obtained with respectto the interface, and v) a photo detecting means capable of detecting anintensity of the light beam, which has been totally reflected from theinterface, with respect to each of the various different angles ofincidence, wherein the improvement comprises:an electrode facing themetal film with the liquid sample intervening therebetween, means forapplying a DC voltage across said electrode and the metal film, a secondprism, said electrode being formed on one surface of said second prismand constituted of a metal film, a second light source for producing asecond light beam, a second optical system for causing said second lightbeam to pass through said second prism and to impinge upon an interfacebetween said second prism and said electrode such that various differentangles of incidence may be obtained with respect to said interfacebetween said second prism and said electrode, and a second photodetecting means capable of detecting an intensity of said second lightbeam, which has been totally reflected from said interface between saidsecond prism and said electrode, with respect to each of the variousdifferent angles of incidence.
 2. A surface plasmon sensor,comprising:i) a prism, ii) a metal film formed on the prism, iii) alight source for producing a light beam, iv) an optical system forcausing the light beam to pass through the prism and impinge upon aninterface between the prism and the metal film such that variousdifferent angels of incidence may be obtained with respect to theinterface, and v) a photo detecting means for detecting an intensity ofthe light beam, which has been totally reflected from the interface,with respect to each of the various different angels of incidence,wherein the improvement comprises:a) an electrode provided on the prismand spaced apart from the metal film, and b) means for applying a DCvoltage across the electrode and the metal film.
 3. A surface plasmonsensor as defined in claim 2, further comprising a layer provided on themetal film, the layer made of a material to which a substance to bedetected specifically adsorbs, such that a state of adsorption isdetectable.
 4. A surface plasmon sensor as defined in claim 2, wherein afirst substance, contained in a sample being analyzed, is immobilized onthe metal film, the first substance being capable of undergoing anantigen-antibody reaction with a second substance, such that a state ofinclusion of a second substance contained in the sample is detectable.5. A surface plasmon sensor as defined in claim 2, wherein the metalfilm and the electrode are formed on one surface of the prism.
 6. Asurface plasmon sensor as defined in claim 5, wherein the metal film andthe electrode are comb shaped, such that the teeth of the metal filmextend into spaces between the teeth of the electrode.
 7. A surfaceplasmon sensor comprising:a prism, a metal film formed on one surface ofsaid prism, a sensor film having fixed thereon one of an antigen and anantibody capable of undergoing an antigen-antibody reaction with asubstance to be analyzed, said sensor film formed on said metal film andbrought into contact with a liquid sample that contains the substance tobe analyzed, a light source for producing a light beam, an opticalsystem for causing the light beam to pass through said prism and toimpinge upon an interface between said prism and said metal film suchthat various different angles of incidence may be obtained with respectto said interface, a photo detecting means capable of detecting anintensity of the light beam, which has been totally reflected from saidinterface, with respect to each of the various different angles ofincidence, an electrode facing said metal film with the liquid sampleintervening therebetween, means for applying a DC voltage across saidelectrode and said metal film, a second prism, said electrode beingformed on one surface of said second prism and constituted of a metalfilm, a second light source for producing a second light beam, a secondoptical system for causing said second light beam to pass through saidsecond prism and to impinge upon an interface between said second prismand said electrode such that various different angles of incidence maybe obtained with respect to said interface between said second prism andsaid electrode, and a second photo detecting means capable of detectingan intensity of said second light beam, which has been totally reflectedfrom said interface between said second prism and said electrode, withrespect to each of the various different angles of incidence.